1. Field of the Invention
The present invention relates to assays for the detection of compounds with pharmacological activity, particularly for the detection of modulators of vascular endothelial growth factor receptor (VEGF-R2) kinase domain.
2. Background
Angiogenesis plays a role in various processes including development of the vasculature, wound healing and maintenance of the female reproductive system. Pathological angiogenesis is associated with disease states such as cancer, diabetic retinopathy, rheumatoid arthritis, endometriosis and psoriasis. The vascular endothelial growth factors (VEGFs) are mediators of both normal and pathologic angiogenesis. VEGF transmits signals into cells through their cognate receptors, which belong to the receptor tyrosine kinase (RTK) family of transmembrane receptors. These receptors are tripartite, consisting of an extracellular ligand-binding domain, a transmembrane domain, which anchors the receptor in the membrane of the cell, and an intracellular tyrosine kinase domain. One subfamily of RTKs comprises the receptors Flt1/VEGF-R1 and KDR/Flk1/VEGF-R2, which bind VEGFs. Binding of the VEGF ligand to the receptor results in stimulation of the receptor tyrosine kinase activity and transduction of biological signals into the cell. The KDR/Flk1/VEGF-R2 receptor mediates the biological activities of mitogenesis and proliferation of endothelial cells while the Flt1/VEGF-R1 receptor mediates functions such as endothelial cell adhesion. Inhibition of KDR/Flk1/VEGF-R2 signalling has been shown to inhibit the process of angiogenesis. Inhibitors of this receptor would be useful in the treatment of diseases where deregulated or uncontrolled angiogenesis exists.
The sequence for the mouse form of VEGF-R2 is described in U.S. Pat. No. 5,283,354. The human sequence is described in U.S. Pat. No. 5,861,301, however, the sequence disclosed in this patent contains several differences from the correct sequence including an inactivating point mutation which renders the protein nonfunctional in the kinase domain. The functional human VEGF-R2 sequence is described in a separate international patent application PCT WO 98/58053. The rat VEGF receptor kinase domain is similar to the corresponding human sequence and was submitted into the public domain on Mar. 13, 1997 (Genbank accession numbers U93306 and U93307) and later described in J. Biol. Chem. 273: 2090–97 (1998). The rat intracellular kinase domain is 97% identical to the human sequence in the N-terminal and C-terminal tyrosine kinase regions, and 89% identical in the intervening Kinase Insert Domain (KID). There is no description of a soluble form of the rat VEGF-R2 kinase domain or the use of such a protein in a method to test compounds for suspected kinase inhibitory activity.
Using scintillation proximity technology, homogeneous assays have been developed for a variety of molecular targets (Cook, N. D. (1996). Drug Discovery Today 1:287–294; Picardo, M. and Hughes, K. T. (1997). In High Throughput Screening [Devlin, J. P. (Ed)], Dekker, New York, N.Y., pp. 307–316). Briefly, the target of interest is immobilized either by coating or incorporation on a solid support that contains a fluorescent material. A radioactive molecule, brought in close proximity to the solid phase by associating with the immobilized target, causes the fluorescent material to become excited and emit visible light. Emission of visible light forms the basis of detection of successful ligand/target interaction, and is measured by an appropriate monitoring device. An example of a scintillation proximity assay is disclosed in U.S. Pat. No. 4,568,649, issued Feb. 4, 1986. U.S. Pat. No. 5,770,176 describes assays for nuclear receptors wherein the functional receptor binds to immobilized nucleic acid. Materials for these types of assays are commercially available from DuPont NEN® (Boston, Mass.) under the trade name FlashPlate™. Development of scintillation proximity assays for the detection of kinase function has been described for purified src tyrosine kinases to peptide substrates (Naykayama, G. R. et al J. Biomolecular Screening (1998) 3:43–48; McDonald, O. B. et al (1999). Anal. Biochem. 268:318–329.) To our knowledge, there are no reported scintillation proximity assays describing soluble VEGF tyrosine kinase function or testing inhibitory compounds using similar techniques.